In this publication, Merck scientists detail the biocatalytic assembly of enlicitide using a suite of enzymes that catalyze selective peptide fragment formation, coupling, and macrocyclization. Together with efficient purifications using crystallization, this strategy enabled the manufacture of a product that would not be possible with traditional synthetic methods. The method described offers a sustainable blueprint for the scalable development of complex macrocyclic peptide therapeutics, environmental advantages and manufacturing efficiencies that support efforts to expand patient access.